This section introduces aspects that may help facilitate a better understanding of the disclosure. Accordingly, these statements are to be read in this light and are not to be understood as admissions about what is or is not prior art.
Motion and change are inherent properties of living systems. The time-scales for quantitatively observing dynamic samples can span microseconds for chemical reactions to milliseconds to seconds for cell and organism motion. Technological advances continue to provide access to new regimes in high-speed imaging capable of capturing full volume renderings at rates previously reserved for single frame acquisitions. High speed cameras with kHz frame rates capable of low-light detection have helped propel new methods for biological microscopy. High frame-rate 3D sectioning capabilities have also been achieved using temporal focusing for two-photon fluorescence and light-sheet microscopy for conventional fluorescence.
Despite these successes, current approaches for high frame-rate imaging using camera-based platforms still suffer from several practical limitations. Firstly, imaging with high frame rates requires high signal to noise ratio (S/N) within each pixel. When detecting sample fluorescence, the high turn-over rates required to produce such S/N can potentially result in significant photobleaching and/or phototoxicity from undesired photochemical reactivity and local heating within the sample. In addition, camera-based imaging is not generally compatible with imaging methods that scale nonlinearly with the incident intensity, including two-photon excited fluorescence (TPEF), second harmonic generation (SHG), coherent anti-Stokes Raman microscopy, and stimulated Raman gain/loss imaging, all of which typically benefit from the higher intensities encountered in beam-scanning instruments. However, one of the most significant limitations of camera-based approaches for high speed biological imaging is the practical difficulties associated with extension to multi-channel detection. Multi-channel detection underpins colocalization experiments, fluorescence resonance energy transfer (FRET) imaging, depolarization ratio detection, and spectral detection. Each channel of detection requires a dedicated high-speed, high sensitivity camera when using the most common high speed microscopy approaches. In addition to the increased complexity and cost of multi-camera detection, precise spatial registry can be challenging to establish and maintain between multiple cameras. In this respect, beam-scanning instruments with multiple single-channel detectors running in parallel offer distinct advantages.
Fast beam-scanning approaches capable of easily supporting multi-channel detection are now well established for video-rate microscopy. Beam-scanning is most commonly achieved by combining a slow-scan galvanometer mirror with a resonant mirror or a rotating polygon mirror for video-rate imaging. However, achieving kHz frame rates using a beam-scanning microscope remains challenging. A 512×512 image contains ˜260,000 pixels, leaving <4 ns per pixel for a 1 ms acquisition to perform the beam positioning and data acquisition. The short duration imposes significant constraints on the beam positioning hardware, the data acquisition electronics, and the sample (e.g., through effects such as saturation, phototoxicity, and mutliphoton absorption, etc.).
There is therefore an unmet need for a high frame-rate imaging technique to resolve the current deficiencies in current imaging techniques as outlined above.